1. Introduction
The microbiota and the microbiome consist of all the conjoint microorganisms and genes that live within the hosts, modulating the health or disease processes. Alterations in microbiota composition are known as dysbiosis [
1]. Understanding the vaginal microbiota is key to effectively preventing urinary tract infections (UTI) [
2].
The presence of commensal microorganisms in the reproductive tract has a role in establishing a fundamental and strong immune state within this particular habitat. One plausible major function of the microbiota in the reproductive tract might be the alteration or limitation of certain constituents within other bacteria in different body regions [
3]. Specific phyla have been identified among the different species and within distinct areas of the female reproductive tract, including the vagina; however, the microbiota’s function in the reproductive tract is not yet fully understood [
3].
Typically, canine vaginal flora comprises a dynamic bacterial population, encompassing both aerobic and anaerobic microorganisms. The bacterial species that are frequently encountered in the canine vaginal tract consist of
Staphylococcus pseudintermedius,
Streptococcus canis,
Enterococcus spp., and
Mycoplasma spp. [
4]. It is worth noting that lactobacilli, which make up the majority of the vaginal microbiota in humans, are rarely isolated from the vaginal tracts of canines. A greater vaginal pH range (5.0 to 8.1) may be a result of the absence of lactobacilli in canines as opposed to humans. Additionally, even in healthy individuals, the presence of bacteria in the vaginal tract of canines, whether unbound or within epithelial cells, is a common occurrence [
2].
The description of the microbiota in the reproductive tract has enabled the detection of subtle changes that may not manifest clinically but might have important clinical significance. Therefore, obtaining a more comprehensive picture of the bacterial microbiome inside the female reproductive system is advantageous, since this knowledge may assist in treating genital tract infections and reproductive failures [
3].
Variation in the vaginal microbiota is observed at various stages of the estrous cycle.
S. canis is more prevalent during proestrus, whereas the presence of
Enterococcus spp. is more frequently observed in dogs with genital tract infections.
Staphylococcus spp. and
Streptococcus spp. have the potential to serve as protective agents against more harmful pathogens through mechanisms such as nutrient competition and epithelial cell receptor adhesion interference [
2,
5]. Studies have shown that a varied bacterial community (including aerobic and anaerobic microbes and opportunistic pathogens) is present in the vaginas of 50% to 100% of clinically healthy dogs [
2]. The typical vaginal microbiota is thought to safeguard the genito-urinary tract against harmful organisms, and diseases in this area are often associated with alterations in the vaginal microflora. Research indicates that bacterial species found in female dogs with reproductive disorders are not considerably different, indicating that infections in the reproductive system may be caused by an excessive expansion of the usual local microbial population [
6]. So far, various publications have researched the vaginal microbiota in different cycle stages, including anestrus or even for spayed (ovariohysterectomy; OHE) bitches [
7,
8,
9,
10]. Moreover, the microbiota of the genital tract (i.e., vagina, cervical, and uterus) of different reproductive cycles [
5,
11] and between healthy and ill bitches with various urogenital problems (i.e., infertility UTI and neonatal mortality) were also assessed [
12,
13,
14,
15,
16].
As information related to the bitch vaginal tract microbiome is quite heterogenous, this study aims to evaluate the prevalence of aerobic bacteria in the vagina according to the lactation period, the type of coitus, seasonality of sampling, age, and reproductive status of gestational and non-gestational bitches.
2. Materials and Methods
2.1. Animals
One hundred lactating bitches between 10 and 168 months of age (45.29 ± 27.62 SD) and weighing between 3 and 65 kg (28.51 ± 27.47 SD) were included in this research study. The bitches were first examined at the Department, and Clinic of Reproduction, Obstetrics and Gynaecology, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania, between January 2013 and December 2014.
In total, 52 (52%) were multiparous, 32 (32%) were primiparous, and 10 (10%) were intact bitches; for the remaining 6, no data were available. Furthermore, 3 bitches (3%) were in the antepartum period (24/48 h before parturition or C-section), 80 (80%) were postpartum (1st week to 6th week after parturition), and 17 (17%) were diagnosed with Lactatio sine graviditate (LSG; 45–60 days after normal estrus when progesterone levels begin to decrease). Regarding mating, 13 bitches (13%) were artificially inseminated, 55 (55%) were naturally controlled reproduced, and 12 (12%) were accidentally reproduced, while, for 20 females, no data were available. Furthermore, 29 samples were collected during spring (March–May), 26 were collected during summer (June–August), 25 were collected during autumn (September–November), and 20 were collected during winter (December–February).
The dogs included in the current study belong to 30 different breeds (including cross breed), namely German Shepherd (n = 21), Cane Corso (n = 9), mongrels (n = 8), Rottweiler (n = 7), Caucasian Shepherd Dog (n = 6), Siberian Husky (=5), Bichon (=4), Dobermann (=4), Yorkshire Terrier (=4), American Staffordshire Terrier (=2), American Bulldog (=2), Beagle (=2), Bucovina Shepherd Dog (=2), Cocker Spaniel (=2), Dachshund (=2), English Bulldog (=2), French Bulldog (=2), German Shorthaired Pointer (=2), Miniature Schnauzer (=2), Pekingese (=2), Saint Bernard (=2), and one Basset Hound, Belgian Shepherd, Boxer, Central Asian Shepherd Dog, Golden Retriever, Labrador retriever, Neapolitan Mastiff, Shih Tzu, and Vizsla from each breed.
The clinical evaluation and diagnostic methodology, including hematological changes, milk and serum acute phase protein levels (APPs), milk cytology evaluation, and milk microbiome results, used to assess the health status of the bitches included in the current study were published by the authors of this research in several other papers [
17,
18,
19]. In total, 40 bitches were healthy; 3 were diagnosed with mammary congestion, 12 with galactostasis, 17 with subclinical mastitis, 12 with acute mastitis, and 1 with gangrenous mastitis, while the other 15, due to lack of data or preliminary laboratory testing, had no definitive diagnostic.
2.2. Sample Collection and Microbiological Analysis
After thoroughly disinfecting the vulvar region with warm water, soap, and ethanol (70%
v/
v), one vaginal swab was taken from each bitch using a sterile bacteriological swab with transport medium (Stuart medium) [
20].
For the microbiological assays, standard microbial methods were used. Thus, bacteriological samples were incubated for 24 h at 370 °C in brain–heart infusion broth (Oxoid, Ltd., Basingstoke, Hampshire, UK). Furthermore, for Staphylococcal growth, the culture was inoculated on Chapman medium agar (Oxoid, Ltd., Basingstoke, Hampshire, UK). Streptococci were grown on blood agar (Oxoid, Ltd., Basingstoke, Hampshire, UK), while Enterobacteriaceae were grown on McConkey medium agar (Oxoid, Ltd., Basingstoke, Hampshire, UK). Finally, the isolates were identified using the Vitek2 identification system (BioMérieux, l’Étoile, France), following the manufacturer’s guidelines.
2.3. Statistical Analysis
The anamnestic data, clinical outcome, and microbiological findings were recorded and saved on an Excel spreadsheet. The data were analyzed using GraphPad Prism version 8.0.0 for Windows, a software developed by GraphPad Software from San Diego, CA, USA. A chi-squared test was used to evaluate the statistical disparities in prevalence. A p-value below the threshold of 0.05 was deemed statistically significant.
4. Discussion
In the current research, 17 bacterial genera were isolated (i.e., 7 G+ and 10 G−). Most G+ genera isolates consisted of
Staphylococcus and
Bacillus, while the G- genera isolates comprised
Proteus,
Agrobacterium, and
Pseudomonas (
Table 1). Associations between G+ and G− bacteria from the same vaginal sample, like
Staphylococcus spp. and
Escherichia coli or
Staphylococcus spp. and
Agrobacterium radiobacter, were frequently encountered (
Table S1).
The cranial vagina of mammals harbors a thriving microbial ecosystem [
5] dominated by Proteobacteria, Bacteroidota, and Firmicutes phyla [
3,
5]. Interestingly, the results of our study are consistent with the current data from the literature.
However, about 60% of the species in the vagina belong to the
Hydrotalea,
Ralstonia,
Fusobacterium, or the
Mycoplasma and
Streptococcus genera [
2,
5,
10,
20,
21]. Nevertheless, concerning these reported data, our study identified only strains from the
Burkholderia genera (i.e.,
Burkholderia cepacia). This result should be carefully interpreted since molecular screening for Mycoplasma and Chlamydia species were not performed.
Furthermore, 82% of our tested vaginal samples were positive for the microbiological culture. These findings are consistent with other reports, where about 71–79% of all the tested bitches had a positive vaginal microbiological culture result [
7].
The literature reports the presence of many bacterial isolates like
Actinomyces coleocanis,
Arcanobacterium pyogenes,
Corynebacterium genitalium,
Enterobacter aerogenes,
Enterococcus avium,
E.
canintestini,
E.
cloacae,
E.
durans,
E.
faecalis,
Erwinia herbicola,
Klebsiella pneumoniae,
Koserella trabulsii,
Obesumbacterium proteus,
Pasteurella multocida,
Proteus mirabilis,
Pseudomonas aeruginosa,
Serratia rubidea,
Sphingomonas paucimobilis,
Staphylococcus aureus, or even
Candida spp. [
7,
8,
9,
11,
14,
15,
22,
23].
To the author’s knowledge, this is the first study where
A. radiobacter,
Ochrobactrum anthropi,
Chromobacterium violaceum, Burkholderia mallei,
Bacillus pumilus,
Streptococcus anginosus,
Streptococcus sanguinis, or
S. xylosus have been isolated from the bitch vaginal secretion (
Table S1). Moreover, literature research shows that the mammalian vagina contains a site-specific microbiota that holds a regulator and/or a drive essential role in many physiological and pathological processes in genital and reproductive health [
24].
However, it is also shown that the vagina is much higher in richness but lower in diversity than the endometrium [
5,
21]. Interestingly, in the present study, the diversity of vaginal strains was richest in the postpartum group compared to the LSG group. The lack of sexual activity for LSG bitches could explain the difference in the population diversity, while, for the postpartum bitches, the opening of the cervix and the puppy passages certainly facilitated the increased diversity of the isolated strains.
Nevertheless, there is significant animal-to-animal variation in the vaginal microbiota [
5], geographical regions influencing the presence of acid-producing bacteria (LAB) [
25], while stray dogs may be potential reservoirs of pathogenic antimicrobial-resistant microorganisms [
23]. Furthermore, age, breed, sex, lifestyle (i.e., urban or rural), or diet influences the core microbiota for different body sites (including the urogenital tract) [
1].
Thus, the prevalence of the vaginal microbiota was affected in the current study by the lactation period (antepartum, postpartum, or LSG), reproductive status (intact, primiparous, or multiparous), type of coitus (naturally controlled, naturally uncontrolled, or artificial insemination), and sampling season (winter, spring, summer, or autumn). The prevalence of the bitch vaginal microbiota has never been assessed using these criteria, as far as the authors are aware.
The vaginal microbiota of bitches in different reproductive phases exhibits a significant level of diversity and variability [
10,
15,
23,
25]. Nevertheless, between intact and OHE bitches, the vaginal microbiome heterogenicity is reduced [
10]. However, in one study, the most present genera in OHE bitches were
Photobacterium (14.03%),
Staphylococcus (11.19%),
Mycoplasma (7.64%), and
Salmonella (11.19%), followed by the Enterobacteriaceae (10.01%) family. In contrast, for anestrus bitches, the most encountered genera were
Mycoplasma (13.90%),
Salmonella (7.60%), and
Staphylococcus (6.80%), followed by the Pasteurellaceae (7.84%) and the Enterobacteriaceae (6.27%) families [
10].
However, a variety in quantity and type of bacteria between different estrous cycle stages [
2] is noticed, especially for females in estrus, where the richness in diversity of the vaginal strains is at the top [
5,
21]. For OHE or infertile bitches,
E. coli,
S. pseudintermedius, and
S. canis are the predominant strains identified [
10,
15,
23,
25].
Our findings are interesting in that, for artificially inseminated, naturally controlled, and uncontrolled coitus bitches, the levels of diversity and variability were similar; for primiparous and multiparous bitches compared to intact ones and for the autumn and winter seasons when compared to spring and summer, the levels of diversity and variability were higher.
Acid-producing bacteria are vaginal residents in the bitch [
7,
26], with only 3% of the isolates belonging to the
Lactobacillus genus, with the majority of isolates corresponding to
Lactococcus spp., followed by
Lactobacillus spp.,
Pediococcus acidilactici,
Lactiplantibacillus plantarum, or
Lactococcus lactis [
26]. Interestingly, no such LABs were isolated in our study due to the use of nonspecific microbiological media.However, in the vaginal vault of spayed bitches, LABs are not predominant [
14].
No significant difference is found between spayed bitches with rUTI and healthy ones. While
E. coli and
S. pseudintermedius are the most often isolated organisms from the vaginal tract of bitches (both healthy and with rUTI),
E. coli tends to be more common in females with rUTI. Moreover,
Enterococcus canintestini was isolated from both healthy and rUTI bitches [
25]. There was comparable prevalence of common vaginal infections among healthy bitches and those who had UTI. Consequently, the incidence of
E. coli is 50% among ill and 42% among healthy bitches, while
S. canis and
S. pseudintermedius have prevalence rates of 30% and 38%, respectively [
2]. Furthermore, in the proestrus phase, for healthy bitches, an increase in
Enterococcus spp. and a decrease in the
E. coli population is noted, with
S. canis being much more common than
Enterococcus spp. [
2].
Some reports suggest that the presence of
Streptococcus spp.,
Lactobacillus spp., or
Enterococcus spp., including
E. canintestini, during proestrus reduces the genital incidence of infections, highlighting a possible protective role for these strains through competitive and adhesion mechanisms for nutrients and the cells’ surface or having potential antimicrobial properties, such as the production of lactic acid, bacteriocin, and hydrogen peroxide, or due to blood irrigation of the vagina during proestrus detrimental to other possible pathogens [
2,
7,
12,
25].
Performing cultures of the vaginal canal will often yield bacterial growth, making it difficult to understand whether therapy is necessary. The process of cultivating bacteria from vaginal swab samples taken from female dogs that do not show any indications of genital illness has limited usefulness. Prior to it, clinical and cytological exams of the vaginal epithelium should always be conducted [
2]. The authors of this research strongly discourage the use of unnecessary or prophylactic antibiotic treatments, as misuse of antimicrobials favors the selection of Methicillin-resistant
Staphylococcus pseudintermedius (MRSP) strains in healthy dogs and their persistence over time [
27].
However, using a broad-spectrum antibiotic doubled by a susceptibility test is mandatory whenever necessary. Noteworthy, a microbiological evaluation of vaginal samples may be used in bitches with a history of fetal mortality. It could be used for selectively treating such females before parturition, especially in cases where β-hemolytic
Streptococcus is isolated [
13].
The presence of phagocytosis and neutrophils on vaginal smears, often indicative of infections, are part of the normal physiological processes observed at the vaginal mucosal surface in bitches. Furthermore, bitches harboring vaginal isolates exhibit a greater fertility rate and produce more robust and healthier offspring compared to bitches without any cultured isolates. These processes are connected with a greater fertility rate [
7].
Finally, there are also reports which show that the same isolates (i.e.,
S. pseudintermedius,
E. coli, or
S. canis) are cultured from both the vagina and the milk secretions during antepartum and postpartum [
16], raising the question of whether or not vaginal bacterial strains are responsible for mammary gland infections, as bacterial translocation mechanisms are acknowledged [
12,
28,
29].
The relatively low number of samples and the lack of molecular identification of isolates represent the main limitations of the current study. Furthermore, future research should focus on anaerobic bacterial cultures in periparturient bitches, to enhance the knowledge variability of bitch vaginal heterogenicity, and the possibility of bacterial translocation from the vagina or myometrium to the lactating mammary glands as a cause for the onset of mastitis.